The present invention relates compounds and methods for the diagnosis and treatment of breast and prostate tumors and metastases thereof, and more particularly to radiolabeled Bombesin peptide analogs for the diagnosis and treatment of breast and prostate tumors and metastases thereof.
Many tumors have biochemical receptors that cause certain molecules, typically peptides or proteins, to bind to the tumor. One approach to diagnose tumors is to identify a compound that binds to a particular tumor and radiolabel the compound with a suitable radionuclide. The labeled compound is then administered the patient, generally via intravenous injection, and allowed to bind to the tumor. The tumor is then located by imaging the location where the radioactive decay occurs. While this concept as presented appears rather simple, in practice it is quite difficult. The first challenge is to identify a candidate compound. If the compound does not bind very strongly to the tumor and for a sufficient period of time, it will not be possible to obtain adequate diagnosis. Further, even if the compound binds to the tumor, if it also binds to surrounding healthy tissue, the diagnosis will be difficult or impossible. Moreover, it is necessary that the linkage of the compound to the radionuclide not disturb the affinity of the tumor for the compound. Another issue is the potential toxicity of the compound in the patient. Compounds that may be very suitable from a binding perspective may be too toxic to use.
A similar approach can be taken in tumor therapy, where one would identify a suitable compound, radiolabel the compound and administer the compound to the patient. The compound is generally administered via intravenous injection, but may to administered by direct injection into the tumor mass. The radionuclide will then decay, releasing energy to kill or reduce the growth of the tumor. Again, the concept is simple, but in practice there are many difficulties. In addition to the problems mentioned above, it is necessary that there be very little of the compound anywhere in the body except in the tumor, due to the danger of the high-energy radiation to healthy tissue. This means that not only must the compound bind very strongly to the tumor, there must be little or no binding to healthy tissues, even if they are not in the vicinity of the tumor.
Attempts to locate suitable compounds have been fraught with difficulty. Because it is desirable to screen large numbers of potential compounds quickly, various “shortcut” assays and models have been developed. Unfortunately, many of these techniques have produced incorrect or misleading data.
Many researchers have used cell line cultures to screen compounds. While the use of cell lines as a screening technique has advantages, it has been found that cell line cultures often have binding affinity for compounds that is not exhibited by actual tumors. Thus the data from this technique produces false positives.
Other researchers have used homogenates of tumors, where a sample of the tumor has been subjected to high shear in a laboratory blender. One problem with this technique is that not only the tumor, but also surrounding tissues that were attached to the tumor are included in the homogenate, thus rendering it impossible to know if any binding affinity is from the tumor or from the surrounding tissues. Further, the shear of the homogenization breaks open the cell membranes, allowing for the possibility of binding that would not occur in an intact cell.
A screening method that produces unambiguous results is a morphological study in which sections (thin slices) of a tumor and surrounding tissue are contacted with a candidate compound that has been labeled with a radionuclide that is suitable for exposing photographic film. This technique clearly differentiates between receptors that are present in the tumor and those present in the surrounding tissue. Unfortunately, this technique is very labor intensive and depends on having suitable tumor tissue samples available.
Peptides and other compounds have been used without radiolabeling to affect the growth of tumors. While some of these compounds may be useful with radiolabeling for imaging and radiotherapy, the correlation between compounds useful for chemotherapy and those useful for radiotherapy is very low.
Bombesin is a peptide originally isolated from frog skin. It is an example of a compound that binds to GRP (Gastrin Releasing Peptide) receptors. Bombesin has the structure:
pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2 (SEQ ID NO: 1)
Considerable work has been conducted in an attempt to identify tumor and non-tumor GRP receptors. Unfortunately, much of that work has yielded results that are inaccurate or misleading.
Breeman et al. “[‘“In-DTPA0 Pro’, Tyr4] bombesin: Studies In Vitro and in Rats”, JNM 39 (1998) 62P teach that high and specific uptake was found in the pancreas and tissues of the GI-tract. Uptake was blocked by iv co-injection of 100 ug of Tyr4-BN with the radiolabeled peptide, but not when administered 1 hour after theradiotracer indicating the internalization of the radioligand.
Hoffman, Li, Sieckman, and Volkert, “Uptake and Retention of a Rh-105 Bombesin Analogue in GRP Receptor Expressing Neoplasms: An In Vitro Study”, JNM 38 (1997) 188P-189P (abstract) teach that the affinity of a Rh-105 labeled bombesin analog for the GRP receptor was investigated along with its prolonged cellular retention in the PC-3 human prostate cancer cell line (65% &commat; 2 h) and CF-PAC1 human pancreatic cell line (41% &commat; 2 h). The Rh-105 analog was rapidly internalized intracellularly in both cell lines studied. The author states that the selective affinity & prolonged retention in neoplastic cells make this radiolabeled peptide a potential candidate for radiotherapy.
Hoffman, Li, Volkert, Sieckman, Higginbotham, and Ochrymowycz, “Synthesis and Characterization of Rh-105 Labelled Bombesin Analogues: Enhancement of GRP Binding Affinity Utilizing Aliphatic Carbon Chain Linkers”, Journal of Labelled Compound and Radiopharmaceuticals 40 (1997) 490-492 (abstract) teach that the IC50 values (using Swiss 3T3 fibroblasts) were determined for a series of 4 peptides and the non-metalated sulfur macrocyclic analogs expressed similar affinities to the GRP receptor than the parent BBN peptide. Upon Rh (III) complexation, decreasing the proximity of the Rh (III) C12-16 and S4 complex to the binding region of BBN, increases the affinity of the final metalated peptide for the GRP receptor. This data may have implications in preparing other metalated BBN analogues which maintain specificity and high affinity for GRP receptors expressed on neoplastic cells.
Hoffman, Sieckman, Ochrymowycz, Higginbotham, Volkert, and Ketring, “In Vitro and In Vivo Characterization of a Rh-105-Tetrathiamacrocycle Conjugate of a Labelled Bombesin Analogue”, JNM 37 (1996) 61P teach that biodistribution studies of the Rh-105 analogue in normal mice showed predominant clearance into the urine and low retention in the kidneys. Data demonstrate the feasibility of forming Rh-105 conjugates with BBN analogs as potential therapeutic agents that specifically target neoplastic cells expressing BBN2 receptors.
Hoffman et al. “Rh-105 Bombesin Analogs: Selective In Vivo Targeting of Prostate Cancer with a Radionuclide” JNM 39 (1998) 982P teach that an Rh-105 BBN (7 14) analog, with a 4 carbon spacer between the sulfur macrocycle and the Q amino acid was evaluated in nude mice possessing PC-3 prostatic tumors. Tumor/Muscle ratios were 7.8, 7.7, and 13.6 &commat; 4, 24, & 48 hours p. i., respectively. The conclusion is that the selective affinity and prolonged retention of this radiolabeled peptide in prostatic cancer cells makes it an attractive candidate for radiotherapy.
T. J. Hoffman, G. L Sieckman, and W. A. Volkert, “Iodinated Bombesin Analogues: Effect of N-terminal vs. Side Chain Iodine Attachment on BBN-GRP Receptor Binding”, JNM 37 (1996) 185P teaches that assessed iodinated BBN analogs as potential SCLC targeting vectors. In all cases, the specific binding region, BBN (8-13) or W-A-V-G-H-L (SEQ ID NO: 2), was maintained, as well as amidation of the carboxy terminal end. Measurement of IC50 values were conducted utilizing Swiss 3T3 cells with [125I] [Tyr4] BBN. The loss of receptor affinity by the mIP-Lys7 conjugated peptide suggests that incorporation of Lys between BBN (1-6) may facilitate increased peptide-receptor affinity. The data show that N-terminal iodination of these analogs may provide a viable route to obtain high affinity BBN iodinated peptides.
Schibli, Hoffman, Volkert. et al. “A Tc-99m DITHIA-DI (Bis-Hydroxymethylene) Phosphine conjugate of Bombesin In Vivo Studies JNM 39 (1998) 225P teaches that the Tc-99 analogs of bombesin derived from 2 different DADT BFCs and the 14 amino acid peptide Lys-3-bombesin were evaluated in a competitive binding assay vs. [125I] [Tyr4] bombesin using human prostate cancer PC-3 cell membranes. The results indicate that the Tc-99m complexes have the potential to be used in the characterization of bombesin/GRP receptors of prostate cancer non-invasively in vivo.
Baidoo et al. “Synthesis and Evaluation of High Affinity Technetium Bombesin Analogs”, JNM 38 (1997) 87P mentions prostate, breast, gastric, colon, pancreatic and scl cancers. DADT chelates (1 and 2, resulting in neutral or positive cores) were attached to the lysine residue & commat; N-terminal region of the potent Bn analog Pyr-Q-K-L-G-N-Q-W-A-V-G-H-L-M-NH2 (SEQ ID NO: 3). When a DADT peptide was labeled with Tc-99m or Tc-99,2 isomers resulted. The Tc-99 analogs exhibited high affinity in a rat cortex membrane binding assay vs. [125I] [Tyr4] bombesin.
B. Rogers, D. Curiel, K. Laffoon, D. Buchsbaum, “Synthesis and Radiolabeling of Bombesin Derivatives with Copper-64 and Binding to Cells Expressing the Gastrin Releasing Peptide Receptor”, Journal of Labelled Compounds and Radiopharmaceuticals 40 (1997) 482 (abstract) concludes that Cu-64-TETA-Aoc BBN (7-14) is a potential therapeutic radiopharmaceutical that can be used to treat GRPr positive tumors.
A. Safavy, M. Khazaeli, H. Qin, and D. Buchsbaum, “Synthesis of Bombesin Analogues for Radiolabeling Rhenium-188”, Cancer 80 (1997) 2354-2359 teaches that 7-amino acid analogue of BBN was synthesized and conjugated to the hydroxamate ligand trisuccin. Radiolabeling with Re-188 were performed in >90% yield. Cell-binding performed with BNR-11 (3T3 mouse fibroblast cells) and PC-3 human prostate carcinoma GRPA positive cells resulted in positive binding.
B. Rogers et al. “Tumor Localization of a Radiolabeled Bombesin Analog in Mice Bearing Human Ovarian Tumors Induced to Express GRP Receptor by an Adenoviral Vector”, Cancer 80 (1997) 2419-2424 shows a study was conducted to determine the level of localization of [125I/131I]-mIP-bombesin in tumors.
Rogers, Buchsbaum, et al. “Localization of I-125-mIP-Des-MetI4-bombesin (7-13) NH2 in Ovarian Carcinoma Induced to Express the GRPr by Adenoviral Vector Mediated Gene Transfer”, JNM 38 (1997) 1221-1229 teaches that [125I] [Tyr4] bombesin was compared to [1211]-mIP-bombesin (a 7 aa BN analog) for in vitro binding and internalization into tumor cells and for tumor localization in vivo, and results showed that the latter has more favorable characteristics with regards to tumor localization and cellular internalization & retention.
Zinn, Buchsbaum, et al. “Imaging Adenoviral-Mediated Gene Transfer of GRPr Using a Tc-99m-Labelled Bombesin Analogue”. JNM 39 (1998) 224P-225P teaches that BBN analogue (QWAVGHLM; SEQ ID NO: 4) was HYNIC modified and radiolabeled with Tc-99m using tricine as a transchelator. Specific and high affinity binding to GRPr- expressing cells was demonstrated by Scatchard analysis. Favorable biodistribution and imaging were observed.
M. E. Rosenfeld et al. Adenoviral Mediated Delivery of GRPr Results in Specific Tumor Localization of a Bombesin Analogue In Vivo”, Clin. Cancer Res. 3 (1997), 1187-1194 teaches similar work to previous publication above.
T. J. Hoffman, G. L. Sieckman and W. A. Volkert. “Targeting Small Cell Lung Cancer Using Iodinated Peptide Analogs” teaches that 5 analogs prepared using SPPS and in vitro BB2 receptor binding assessed using Swiss T3T fibroblasts. Amino acids #1-6 nor C-terminal Met residue are not essential to maintain receptor specificity. Results imply that incorporation of I-123 or I-131 as a m-iodophenyl moiety may be used to diagnose or treat sclc.
U.S. Pat. No. 5,686,410 Novartis Albert teaches radiolabeled bombesin and antagonists, including use for tumor imaging and therapy (Examples 11 and 12).
There are numerous articles and patents that discuss the binding of non-radioactive bombesin analogs to various tissues such as SCLC (small cell lung cancer), and pituitary, adrenal, and skin tumors.